Bacteria and their toxins have been investigated for their anticancer activities. In the 1970s, bacteria (such as non-pathogenic Clostridium) were used for the treatment of malignant brain tumors, but the tumors recurred in these brain tumor patients. More than 100 microorganisms have been studied for their potential anticancer activities, and many bacteria have growth specificity for tumors that is 1000 times greater than for other tissue.
While their anti-tumor activities make many bacteria attractive therapeutic agents, there are inherent risks to administering live bacteria to humans. A safer and more effective strategy has been to use biological toxins, specifically from bacteria, as therapeutic agents. Bacterial toxins are not only toxic, but are also highly specific for certain cell types, or can be engineered to be specific by fusing the toxin to other molecules. Many bacterial toxins are able to enter mammalian cells where they exert their toxic effects. Because of extensive evolutionary adaptation between bacteria and their hosts, bacteria have become very good at “developing” highly effective toxins.
Each year, more than 60,500 people die of hematologic malignancies (leukemia, lymphoma, myeloma) with more than 110,000 new annual diagnoses in the US alone. Current treatment for these cancers includes the use of synthetic compounds that target the cell division process of nearly all cells of the body, not just the cancerous ones. As a result, devastating side effects are all too common. Furthermore, a significant percentage of patients eventually show resistance to many of the drugs, thus rendering treatment largely ineffective. Indeed, there is an effort to identify agents that induce cancer cell death by methods other than damage to DNA or cell division.
While the drugs currently in use are toxic for cells, they are not highly specific. A new class of therapeutic agents for the treatment of hematologic malignancies, and cancer in general, includes drugs that exhibit specificity for predominantly the cancerous cell type. Examples of targeted therapeutics include Rituximab, which is a monoclonal antibody against B-lymphocytes, and Mylotarg, an antibody-anti-tumor antibiotic fusion directed against cells of myelomonocytic lineage.
Actinobacillus actinomycetemcomitans is a Gram negative pathogen that inhabits the oral cavities of humans. A. actinomycetemcomitans is the etiologic agent of localized aggressive periodontitis (LAP), a rapidly progressing and destructive disease of the gingiva and periodontal ligaments. Among its many virulence factors, A. actinomycetemcomitans produces an RTX (repeats in toxin) leukotoxin. A. actinomycetemcomitans leukotoxin is an approximately 115 kDa protein that kills specifically leukocytes of humans and Old World Primates. Leukotoxin is part of the RTX family that includes E. Coli α-hemolysin (HlyA) and Bordetella pertussis adenylate cyclase (CyaA). Leukotoxin may play an important role in A. actinomycetemcomitans pathogenesis by helping the bacterium destroy gingival crevice polymorphonuclear leukocytes (PMNs) and monocytes, resulting in the suppression of local immune defenses.
The initial identification and testing of novel anti-cancer agents relies on in vitro killing assays using relevant cancer cell lines. While in vitro assays performed under cell culture conditions prove useful and necessary for preclinical testing of new therapeutics, extrapolation to the physiological conditions of a living organism is often difficult or impossible (27). Because of the high cost of drug development ($800 million), new drug screens are constantly being sought to more efficiently eliminate or identify candidate therapeutic agents (27). Indeed, increasing the clinical success rate from ⅕ to ⅓ because of more effective preclinical drug screens would reduce drug development costs by more than $200 million (27).
The activity, specificity, or toxicity of a compound in the physiological environment can vary significantly from cell culture conditions. While no in vitro assay or screen can represent the complexity of the human body, several assays have been developed to more closely mimic in vivo conditions. Several of these assays include the colony forming cell assay using bone marrow cells (27,29), hepatic drug biotransformation assays (3), and assays in whole blood (4,45). Because most chemotherapeutic agents are administered intravenously and are therefore immediately affected by blood cell components, screening for potential drugs in the presence of whole blood would be expected to yield more meaningful results. Blood contains biological components, such as proteases, antibody, and blood cells, which can affect the nature of a compound. For example, red blood cells and plasma proteins are known to affect the pharmacokinetics of drugs such as the anti-cancer agents docetaxel and gemcitabine (8,9). Vaidyanathan et al. (43) also reported that the cardioprotective drug, dexrazoxane, inhibits binding of the anti-cancer agent, doxorubicin, to red blood cells and that this interaction alters the pharmacokinetics of doxorubicin, and Clarke et al. (4) used an in vitro whole blood assay to study the binding affinity of a surrogate anti-CD11a monoclonal antibody to blood components. In addition, leukocytes produce a cytochrome P450 isoform (CYP2E1) that is involved in drug biotransformation (3). Thus, identifying and studying drugs in the presence of whole blood or blood components can offer a unique advantage over assays using cells in monoculture.
For studies on leukemia therapeutics, the cell line HL-60 is used as a standard target cell line. HL-60 cells were originally isolated from a 36-year-old female patient with acute promyelocytic leukemia (13). Testing the efficacy of anti-leukemia therapeutics against HL-60 cells in whole blood or other biological material is currently a challenge due to the inefficiency in differentiating the viability of HL-60 cells from other cells.
Thus, there remains a need for the identification and development of therapeutic agents and strategies, for the treatment of cancers such as leukemia, and for the development of effective testing methodology, such as efficient screens for therapeutics such as anti-leukemia agents, and particularly, for the facilitation of preclinical studies on a highly specific bacterial leukotoxin as a novel anti-leukemia therapeutic agent.